Numerous reagents have been used to attach biotin to proteins via thiol and amino functions. Examples of non-cleavable biotinylating reagents specific for the thiol function include 3-(N-maleimido) propionyl biocytin, described by Bayer et al, Analytical Biochemistry, 149, 599-536 (1985), and N-iodoacetyl-N',-biotinylhexylenediamine described by Sutoh et al, J. Mol.Biology, 178, 323-339 (1984). Examples of cleavable biotinylating reagents specific for the amino function include 3-(4-(N-biotinoyl -6-aminocaproyloxy) phenyl) propionic acid N-hydroxysuccinimide ester (BPE) described by Mouton et al, Archives of Biochemistry and Biophysics, 218, 101-108 (1982) and sulfosuccinimidyl 2-(biotinamido) ethyl 1,3'-dithiopropionate (NHS-SS-Biotin) sold by Pierce Chemical Company.
For some applications, it is desirable to remove the biotin residue and regenerate free, unlabeled protein. For example, the biotin residue may be employed together with immobilized avidin for the retrieval of a thiol containing protein from a mixture containing same. Once isolated, it may be important to study the protein in the unmodified state. In such cases, it is necessary to cleave the biotin residue under conditions which do not affect the integrity of the protein.
Those biotinylating reagents described above which are cleavable, i.e. BPE and NHS-SS-Biotin, leave a portion of the reagent, namely an alkylamido residue, on the protein. Since many proteins contain free thiol functions and/or disulfide functins which are readily reduced to free thiol functions, a cleavable biotinylating reagent specific for thiol functions which allows regeneration of free, unlabeled protein would have broad applicability in affinity purification techniques.